The efficient production of bioactive proteins and peptides has become a hallmark of the biomedical and industrial biochemical industry. Bioactive peptides and proteins are used as curative agents in a variety of diseases such as diabetes (insulin), viral infections and leukemia (interferon), diseases of the immune system (interleukins), and red blood cell deficiencies (erythropoietin) to name a few. Additionally, large quantities of proteins and peptides are needed for various industrial applications including, for example, the pulp and paper industries, textiles, food industries, personal care and cosmetics industries, sugar refining, wastewater treatment, production of alcoholic beverages and as catalysts for the generation of new pharmaceuticals.
In some cases commercially-useful proteins and peptides may be synthetically produced or isolated from natural sources. However, these methods are often expensive, time consuming, and characterized by limited production capacity. Recombinant microbial production may be use to commercially produce the desired peptide/protein. Although preferable to synthesis or isolation, recombinant expression of peptides has a number of obstacles to be overcome in order to be a cost-effective means of production. For example, peptides (and in particular short peptides) produced in a cellular environment are susceptible to degradation from the action of native cellular proteases. Additionally, purification can be difficult, resulting in poor yields depending on the nature of the protein or peptide of interest.
One means to mitigate the above difficulties is the use of a genetic chimera for protein and peptide expression. A chimeric protein or “fusion protein” is a polypeptide comprising at least one portion of the desired protein product fused to at least one portion comprising a peptide tag. The peptide tag may be used to assist protein folding, assist post expression purification, protect the protein from the action of degradative enzymes, and/or assist the protein in passing through the cell membrane.
In many cases it is useful to express a peptide in an insoluble form, particularly when the peptide of interest is rather short, soluble, and/or subject to proteolytic degradation within the host cell. Production of the peptide in insoluble form both facilitates simple recovery and protects the peptide from the undesirable proteolytic degradation. One means to produce the peptide in insoluble form is to recombinantly produce the peptide as part of an insoluble fusion peptide by including within the fusion peptide at least one peptidic solubility tag (i.e., an “inclusion body tag” or “IBT”) that induces inclusion body formation. Typically, the fusion peptide is designed to include at least one cleavable peptide linker separating the inclusion body tag from the peptide of interest so that the peptide of interest can be subsequently recovered from the fusion peptide. The fusion peptide may be designed to include a plurality of inclusion body tags, cleavable peptide linkers, and regions encoding the peptide of interest.
Fusion proteins comprising a peptide tag that facilitate the expression of insoluble proteins are well known in the art. The solubility tag of the chimeric or fusion protein is often large, increasing the likelihood that the fusion protein will be insoluble. Example of large peptide solubility tags include, but are not limited to chloramphenicol acetyltransferase (Dykes et al., Eur. J. Biochem., 174:411 (1988), β-galactosidase (Schellenberger et al., Int. J. Peptide Protein Res., 41:326 (1993); Shen et al., Proc. Nat. Acad. Sci. USA 281:4627 (1984); and Kempe et al., Gene, 39:239 (1985)), glutathione-S-transferase (Ray et al., Bio/Technology, 11:64 (1993) and Hancock et al. (WO94/04688)), the N-terminus of L-ribulokinase (U.S. Pat. No. 5,206,154 and Lai et al., Antimicrob. Agents & Chemo., 37:1614 (1993), bacteriophage T4 gp55 protein (Gramm et al., Bio/Technology, 12:1017 (1994), bacterial ketosteroid isomerase protein (Kuliopulos et al., J. Am. Chem. Soc. 116:4599 (1994), ubiquitin (Pilon et al., Biotechnol. Prog. 13:374-79 (1997), bovine prochymosin (Naught et al., Biotechnol. Bioengin. 57:55-61 (1998), and bactericidal/permeability-increasing protein (“BPI”; U.S. Pat. No. 6,242,219 to Better et al.). The art is replete with specific examples of this technology, see for example U.S. Pat. No. 6,613,548, describing fusion protein of proteinaceous tag and a soluble protein and subsequent purification from cell lysate; U.S. Pat. No. 6,037,145, teaching a tag that protects the expressed chimeric protein from a specific protease; U.S. Pat. No. 5,648,244, teaching the synthesis of a fusion protein having a tag and a cleavable linker for facile purification of the desired protein; and U.S. Pat. Nos. 5,215,896; 5,302,526; 5,330,902; and 7,501,484, describing fusion tags containing amino acid compositions specifically designed to increase insolubility of the chimeric protein or peptide.
Shorter inclusion body tags have been developed from the Zea mays zein protein (U.S. Pat. No. 7,732,569), the Daucus carota cystatin (U.S. Pat. No. 7,662,913), an amyloid-like hypothetical protein from Caenorhabditis elegans (U.S. Pat. Nos. 7,427,656 and 7,795,382), and a ketosteroid isomerase-derived solubility tag modified to be more acid resistant (U.S. Pat. No. 7,829,311 to DeCarolis et al.). The use of short inclusion body tags increases the yield of the target peptide produced within the recombinant host cell.
U.S. Patent Application Publication No. 2009-0043075A1 to Alsop et al. discloses fusion proteins comprising cross-linkable inclusion body tags. After cleaving the recovered fusion protein, oxidative cross-linking is used to separate the inclusion body tag from the polypeptide of interest.
U.S. Pat. Nos. 7,678,883 and 7,794,979 to Cheng et al. disclose inclusion body tags derived from an 11 amino acid synthetic peptide (i.e. peptide “PII-2”; also known as peptide “DN1”) capable of self-assembly into β-sheet tapes, ribbons, fibrils, and fibers in water has been described (Aggeli et al., J. Amer. Chem. Soc., 125:9619-9628 (2003); Aggeli et al., PNAS, 98(21):11857-11862 (2001); Aggeli et al., Nature, 386:259-262 (1997); and Aggeli et al., J. Mater Chem, 7(7):1135-1145 (1997).
Fusion peptides comprising a solubility tag are typically subjected to a cleavage step to separate the solubility tag from the peptide of interest. Separating the solubility tag from the peptide of interest may be particularly desirable if the presence of the tag adversely impacts the properties of the peptide of interest. However, cleavage of the fusion peptide followed by one or more purification steps adds significant cost to the overall process.
One way to reduce the cost of producing a fusion peptide comprising a solubility tag is to use an inclusion body tag that does not need to be removed during downstream processing. Preferably, the “leave on” solubility tag is capable of providing additional functionality (beyond inclusion body formation) to the peptide of interest. Such multi-functional “leave on” solubility tags would significantly reduce the cost of manufacture and provide a higher value product
Small peptides may be used in material science applications based on their ability to bind to a target material. These “target surface-binding peptides” may be used to prepare peptide-based reagents (e.g., peptides of interest) designed to couple or deliver a benefit agent to the target material. Peptide-based reagents have been used to target cosmetic benefit agents to a variety of body surfaces (U.S. Pat. Nos. 7,220,405; 7,309,482; 7,285,264 and 7,807,141; U.S. Patent Application Publication Nos. 2005-0226839 A1; 2007-0196305 A1; 2006-0199206 A1; 2007-0065387 A1; 2008-0107614 A1; 2007-0110686 A1; 2006-0073111 A1; 2010-0158846; 2010-0158847; and 2010-0247589; and published PCT applications WO2008/054746; WO2004/048399; and WO2008/073368).
The problem to be solved is to provide a solubility tag that is (1) effective in preparing fusion proteins which accumulate in an insoluble form within the host cell (i.e., forming inclusion bodies), and (2) does not need to be cleaved from the fusion proteins prior to the intended use (i.e., the presence of the solubility tag in the fusion peptide/protein does not adversely impact the desired functionality of the polypeptide/protein of interest). In a preferred embodiment of the problem to be solved, the solubility tag is not removed and also provides an additional beneficial property (in addition to driving the formation of inclusion bodies) to the fusion peptide comprising a peptide of interest. Preferably the additional or enhanced property is the ability to control and/or enhance deposition and/or the binding properties of the fusion peptide for the desired target surface.